R: qPCR Experiment for the Amplification of MLC-2v Using the...
C127EGHP
R Documentation
qPCR Experiment for the Amplification of MLC-2v Using the Roche Light Cycler
1.5
Description
Quantitative PCR (qPCR) with a hydrolysis probe (Cy5/BHQ2) and DNA binding dye
(EvaGreen) (Mao et al. 2007) was performed in the Roche Light Cycler 1.5
thermo cycler. The cycle-dependent increase of the fluorescence was
quantified at the annealing step.
Usage
data(C127EGHP)
Format
A data frame with 40 observations on the following 66 variables. The first columns
("index") contains index of a sample and second column ("Cycle") contains the number
of cycle. Consecutive columns EG1-EG32 contains fluorescence data for Eva Green dye.
Consecutive columns HP1-HP32 contains data for hydrolysis probe.
Details
MLC-2v was amplified in the Roche Light Cycler 1.5. The the change of
fluorescence was simultaneously monitored for the Hydrolysis probe of MLC-2v
and EvaGreen. The primer sequences for MLC-2v were taken from Roediger et al.
(2013). A 10 micro L qPCR reaction was composed of 250 nM primer (forward and
reverse), qPCR Mix (according to the manufactures recommendations), 1 micro L
template (MLC-2v amplification product), 60 nM hydrolysis probe probe for
MLC-2v. EvaGreen was used at 0.5 x final. During the amplification was
monitored 59.5 degrees Celsius.
A Highly Versatile Microscope Imaging Technology Platform for the Multiplex
Real-Time Detection of Biomolecules and Autoimmune Antibodies. S. Roediger,
P. Schierack, A. Boehm, J. Nitschke, I. Berger, U. Froemmel, C. Schmidt,
M. Ruhland, I. Schimke, D. Roggenbuck, W. Lehmann and C. Schroeder.
Advances in Biochemical Bioengineering/Biotechnology. 133:33–74, 2013.
http://www.ncbi.nlm.nih.gov/pubmed/22437246
Mao, F., Leung, W.-Y., Xin, X., 2007. Characterization of EvaGreen and the
implication of its physicochemical properties for qPCR applications.
BMC Biotechnol. 7, 76.
Examples
str(C127EGHP)
data(C127EGHP)
tmp <- C127EGHP
par(mfrow = c(2,1))
plot(NA, NA, xlim = c(1,40), ylim = c(0,10), xlab = "Cycle",
ylab = "Fluorescence", main = "MLC-2v qPCR - EvaGreen")
for (i in 3:34) {
points(tmp[, 2], tmp[, i], type = "l", col = i)
}
plot(NA, NA, xlim = c(1,40), ylim = c(0,10), xlab = "Cycle",
ylab = "Fluorescence", main = "MLC-2v qPCR - Hydrolysis probe")
for (i in 35:66) {
points(tmp[, 2], tmp[, i], type = "l", col = i)
}
par(mfrow = c(1,1))